1. Field of the Invention
This invention generally relates to human lipid metabolism, particularly to HDL-related proteins, their mutations, and peptides designed based on these mutations which have antioxidant properties beneficial in the regulation of cardiovascular disease (CVD), bone diseases and other inflammatory related diseases.
2. Description of the Related Art
Cardiovascular disease (CVD) is the number one cause of death in Western societies and its prevalence is increasing worldwide. One of the strongest predictors of risk is the plasma concentration of high density lipoprotein (HDL) which exhibits an inverse relationship to the risk (Gordon, T., et al., Am. J. Med. 62:707–714, 1997; Wilson, P. W. F. , Am. J. Cardiol. 66:7A10A, 1990). Despite the strong epidemiological data relating increased plasma HDL to protection against CVD, a number of rare inheritable traits have been described which result in low plasma HDL concentrations but no increase in CVD. These inheritable traits are, in part, attributed to mutations in apolipoproteinA-I, the major protein component of HDL (Assmann, G., et al, Circulation 87:[suppl III]:III-28-III-34, 1993).
ApolipoproteinA-IMilano and apoA-IParis are examples of natural variants of apoA-I that manifest HDL deficiencies but there is no apparent CVD in affected subjects. See Weisgraber, K. H., et al., J. Clin. Invest. 66:901–907, 1980; Franceschini, G., et al., i J. Clin. Invest. 66:892–900, 1980; Bruckert, E., et al., Atherosclerosis, 128:121–128, 1997. Indeed, a recent clinical study showed that carriers of apoA-IMilano exhibited normal intimal thickness of carotid arteries compared to age- and sex-matched controls; whereas, hypoalphalipoproteinemic individuals showed intimal thickening as judged by B-mode ultrasound (Sitori, C. R., et al., Circulation 103:1949–1954, 2001). Studies utilizing mice and rabbits support clinical studies by demonstrating that injection of recombinant apoA-IMilano protects against atherosclerosis (Shah, P. K., et al., Circulation 97:780–785, 1998; Shah, P. K., et al., Circulation 103:3047–3050, 2001; Ameli, S., et al., Circulation 90:1935–1941, 1994). However, the mechanism(s) by which apoA-IMilano and apoA-IParis exert anti-atherogenic effects are not completely understood.
All known human carriers of apoA-IMilano and apoA-IParis are heterozygous for R173C and R151C mutations in apoA-I primary sequence, respectively (Weisgraber, K. H., et al., J. Clin. Invest. 66:901–907, 1980; Bruckert, E., et al., Atherosclerosis, 128:121–128, 1997). The introduction of a cysteine residue in a normally cysteine-free apolipoprotein allows for the formation of homodimers and heterodimers with apoA-II. Dimerization of the cysteine variants inhibits HDL maturation via mechanisms related, in part, to impaired activation of lecithin: cholesterol acyltransferase, the enzyme that catalyzes cholesterol esterification on HDL (Franceschini, G., et al., J. Biol Chem. 265:12224–12231, 1990; Calabresi, L., et al., Biochem. Biophys. Res. Comm. 232:345–349, 1997; Daum, U., et al., J. Mol. Med. 77:614–622, 1999). ApoA-IMilano apoA-IParis are rapidly cleared from the plasma compartment in humans thus contributing to the HDL deficiency in vivo (Roma P, et al., J. Clin. Invest. 91:1445–1452, 1993; Perez-Mendez, O., et al., Atherosclerosis 148:317–326, 2000). However, the fractional catabolic rate of apoA-IParis appears to be different from that of apoA-IMilano suggesting that the two cysteine variants may differ in their metabolic behavior. Human carriers of apoA-IMilano and apoA-IParis also exhibit mild hypertriglyceridemia in addition to the HDL deficiency (Bruckert, E., et al., Atherosclerosis, 128:121–128, 1997; Franceschini G., et al., Atherosclerosis 7:426–435, 1987).
The C-terminal lipid-binding domain of Apo A-IWT consists of a series of helical repeats separated by proline residues. The amphipathic alpha helix 7 containing R173C is flanked by two amphipathic alpha helices of relatively greater lipid binding affinity. The lipid binding affinity of the helical repeats alternate, but the two end helices of Apo A-I exhibit the highest lipid-binding affinity (Palgunachari, M.N., et al., Arterioscier. Thromb. Vasc. Biol. 16:328–338, 1996). The relatively low lipid-binding affinity associated with helix 7, where R173C is located, may allow a high degree of movement of this particular helix on phospholipid surfaces thus maximizing the freQuency of collision between the free thiol at position 173 with reactive lipid peroxides. Increased flexibility of helix 7, which is located in the central region of the C-terminal lipid-binding domain, may be optimized in the presence of deoxycholate used in the preparation of the phospholipid micelles.
The paradox of abnormal lipoprotein metabolism and protection from CVD has led to the suggestion that the cysteine substitution for arginine in the lipid-binding domain of apoA-I may impart a gain-of-function protecting against atherosclerosis. As thiol groups in proteins are strong nucleophiles often participating in electron transfer reactions, we hypothesized that the monomeric forms of apoA-IMilano and apoA-IParis which contain a free thiol, may possess an antioxidant activity distinct from that of apoA-IWT.
Individuals with these substitutions are known to have low levels of the “good” cholesterol HDL, but yet do not suffer from significantly increased levels of CVD. Oda et al. disclose cysteine substitutions in Apolipoprotein A-I in Biochemistry 40 (2001) 1710–1718 other substitutions are disclosed at Atherosclerosis 128 (1997) 121–128 ; Atherosclerosis 135 (1997) 181–185. Antioxidant action of HDL is discussed at Atherosclerosis 135 (1997) 193–204.
These cysteine for arginine substitutions in the Apo A-I variant is of special interest in treatment of cardiovascular disease. The dimer of Apolipoprotein apoA-IMilano and the process of producing and purifying the dimer composition have been disclosed by Sirtori et al, in U.S. Pat. No. 5,876,968, which is hereby incorporated by reference. The process described by Sirtori et al. relies on converting any monomer present to a substantially pure form of the dimer form of apoA-IMilano of at least 90% purity.
Segrest et al., in U.S. Pat. No. 4,643,988, discloses amphipathic peptides that are useful for treatment and prevention of athersclerosis. The Segrest peptides, generally referred to as 18A and 18pA, are based on an idealistic model of an amphipathic alpha helix that possesses a primary amino acid sequence distinct from that of apoA-I. However, the peptides form Class A amphipathic alpha helices with positively charged amino acids at the interface of polar/nonpolar region and negatively charged residues located in the middle of the polar face of the helix.
Segrest et. al. described the use and properties of 18A and 18pA; the latter representing a series of two 18A peptides linked by a proline residue. The sequence of 18A is as follows: DWLKAFYDKVAEKLKEAF (SEQ ID NO:75). Various conservative substitutions (for example positively charged lysine residues in place of positively charged argine residues) that ndo not change the overall design of the class A amphipathic alpha helix are also claimed. Additional substitutins of D- for L- amino acid isoforms are described as well as replacement of naturally ocurring amino acids for synthethic derivatives (i.e. substitutions of alanine for alapha-naphthylalanine). While an amphipathic peptide is disclosed, the peptide does not possess a cysteine residue and thus lacks the antioxidant activity shown to be possessed by apoA-IMilano.
Garber et al., disclose on U.S. Pat. No. 6,156,727, anti-atherosclerotic peptides and a transgenic mouse model of atherosclerosis. Garber et al. utilize the same peptides as described above by Segrest et al., however Garber et al. created transgenic mice that express the peptides 18A and 37pA, the latter sometimes is referred to as 18A-Pro-18A. Again, these peptide does not possess a cysteine residue and thus lack the antioxidant activity shown to be possessed by apoA-IMilano.
Lees et al., disclose in U.S. Pat. No. 5,955,055, synthetic peptides for arterial imaging at vascular imaging sites, that mimic apolipoprotein B (apoB), apolipoprotein A-I or elastin proteins and is hereby incorporated by reference in its entirety. The Lees peptides are derived (mostly) from apoB and elastin/collagen and are not similar to the peptides we now disclose. The following sequence is used as is based on apoB: YRALVDTLKFVTQAEGAL (SEQ ID NO:89). The sequence derived from apoA-I described by Lees et al. is: YVLDEFREKLNEELEALKQ (SEQ ID NO:90). There is no exact sequence match to apoA-I, probably because of conservative substitutions, and the peptide is not at all similar to any of the peptides we now disclose.
Moreover, none of the peptides in the above mentioned patents are based on Apolipoprotein E3 (apoE3) and Apolipoprotein A-V (apoAV). This is because the mechanisms responsible for the antioxidant properties of apoE3 have not been fully defined until now. ApoAV is a new apolipoprotein that has recently been described and very little is known about its function. Thus, peptides based on apoAV provide new avenues for development of therapeutic agents. It is also clear from our studies that the antioxidant properties of apoA-IMilano and its peptide mimetics are specifically directed toward phospholipid surfaces which none of these above-mentioned patented peptides are shown to be directed toward.